Halofuginone does not reduce fibrosis in bleomycin-induced lung injury

Halofuginone, a coccidiostatic alkaloid, has anti-fibrotic properties, and may be useful as a therapeutic agent in lung fibrosis. To test this hypothesis we investigated the effect of halofuginone on bleomycin-induced lung fibrosis in Sprague-Dawley rats. Treatment groups included: (1) a single intratracheal (IT) instillation of 1.2U bleomycin, and intraperitoneal (IP) injection of halofuginone (0.5 mg/dose), every other day; (2) IT 1.2U bleomycin and IP distilled water (D.W.), every other day; (3) IT 0.8U bleomycin and daily IP halofuginone (0.5 mg/dose); (4) IT 0.8U bleomycin and daily IP D.W.; (5) IT saline and IP halofuginone, every other day; (6) IT saline and daily IP D.W.; (7) IT 0.625U bleomycin and oral halofuginone (10 mg/kg rodent lab chow); (8) IT 0.625U bleomycin and standard lab chow. Animals were studied 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semi-quantitative morphological index of lung injury, and by biochemical analysis of lung hydroxyproline content. Overt signs of lung injury were apparent in bleomycin-treated rats by all measures. These changes were not affected by treatment with halofuginone, irrespective of the treatment regimen used. This study does not support the use of halofuginone to prevent or ameliorate lung fibrosis.     

Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment

Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody’s assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.     

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of halofuginone in plasma

An LC-MS/MS method was developed to quantitate the potential antitumor agent halofuginone in plasma. The assay uses 0.2 ml of plasma; chlorohalofuginone internal standard; acetonitrile for protein precipitation; a Phenomenex SYNERGI 4 micro Polar RP 80A (4 microm, 100 mm x 2 mm) column; an isocratic mobile phase of methanol:water:formic acid (80:20:0.02, v/v/v); and positive-ion electrospray ionization with selective reaction monitoring detection. Halofuginone eluted at approximately 2.4 min, internal standard eluted at approximately 2.9 min, and no endogenous materials interfered with their measurement. The assay was accurate, precise, and linear between 0.1 and 100 ng/ml. Halofuginone could be quantitated in dog plasma for at least 24 h after an i.v. dose of 0.1mg/kg. The assay is being used in ongoing pharmacokinetic studies of halofuginone.     

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.     

Chemical synthesis of febrifugine and analogues

The quinazolinone-containing 2,3-disubstituted piperidines febrifugine and isofebrifugine have been the subject of significant research efforts since their occurrence in Dichroa febrifuga and their anti-malarial actions were first described in the late 1940s. Subsequently they have also been shown to be present in other plants belonging to the hydrangea family and various analogues of febrifugine have been prepared in attempts to tune biological properties. The most notable analogue is termed halofuginone and a substantial body of work now demonstrates that this compound possesses potent human disease relevant activities. This review focuses on the literature associated with efforts dedicated towards uncovering the structures of febrifugine and isofebrifugine, the development of practical methods for their synthesis and the syntheses of structural analogues.     

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy

Transforming growth factor-β (TGF-β) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-β₁ signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-β₁ in cultured mesangial cells. In addition, the expression of TGF-β type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-β₁. An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.